DESCRIPTION: (adapted from applicant?s abstract) The goal of the proposed project is to better understand the fundamental aspects of the relationship between the prosthetic group of a protein with its surrounding polypeptide chain. This will be done using a combination of computational predictions, synthetic skills and unique spectroscopic probes that are now available. The proposed studies will focus on prosthetic groups (in heme proteins) on protein residues and protein co-factors (in Ca-binding proteins) that have spectroscopic properties sensitive to the conformational motions of the protein, to the electrostatic environment and to subtle structural details. These properties will be used as indicators of important chemical and physical protein features under a variety of experimental conditions. The proposed studies will build on work done during the previous granting period. Specific infrared markers that can provide information on active sites (metal ligands), overall protein folding (amide vibrations), protein surface phenomena (carboxyl and SH groups) and on the water interface (OH) have been characterized for cytochrome-c and parvalbumin. In this period, vibrational spectroscopy combined with molecular dynamics simulations and continuum electrostatic calculations will be used to study the effect of the protein electric field on these new direct spectroscopic probes. This project is unique in that it supplements precisely determined optical and vibrational markers with a molecular dynamics/electrostatic potential approach allowing us to explore how a protein might adjust its structural stability to perform its physiological function. The specific aims are indicated as follows: 1) To focus on prosthetic groups (in heme proteins) and on protein-residues and on protein cofactors (in Ca binding proteins) that have spectroscopic properties sensitive to the conformational motions of the protein, the electrostatic environment and to subtle structural details. 2) Electrostatic calculations and pH-dependence studies will be used to study the role of charged groups in creating and modulating protein electrostatic potentials. Relative importance of the protein matrix and the heme ligands on the electronic and vibrational properties of the heme will be studied. 3) The extent of conformational fluctuations of a protein under native, mildly denaturing, and strongly denaturing conditions (and how these fluctuations affect the heme properties) will be studied making use of available single molecule detection facilities (Hochstrasser). Spectral shift analysis of Zn cytochrome-c will be used to examine the dynamics of the protein over a long period of time (Hochstrasser) and the results will be correlated with molecular simulations (Sharp). 4) The relaxation of a protein after a perturbation making use of the binding and release of Ca from parvalbumin and calmodulin. The focus of these studies will be on the dynamical behavior observed by sampling through varying solution conformations. Temperature dependent folding and unfolding events will also be studied.